Methods of sequencing with linked fragments

ABSTRACT

The invention generally relates to sequencing library preparation methods. In certain embodiments, two template nucleic acids are joined together by a linking molecule, such as a PEG derivative. The linked template nucleic acids is amplified, creating linked amplicons.

FIELD OF THE INVENTION

The invention generally relates to methods of amplifying and sequencingnucleic acids.

BACKGROUND

High-throughput genomic sequencing platforms generate large amounts ofdata at affordable prices, but they are not sufficiently accurate. Eventhe best sequencing techniques have error rates around 1 percent. Thattranslates to hundreds of thousands of errors in the sequence of asingle human genome. Inaccurate base calling leads to sequencemisalignment and the misidentification of mutations. Although basecalling and alignment algorithms are available, quality is negativelyimpacted by amplification and sequencing errors.

Although advances have been made in amplification and sequencingtechniques, base calling and alignment remain riddled with errors. Forexample, in the currently leading sequencing platform, DNA fragments areattached to a solid support, such as a channel wall. Once a fragment isattached to the solid support, the fragment is amplified and theamplification products attach to the solid support proximate to theseeding fragment. The process repeats until a cluster of amplificationproducts identical to the seeding fragment forms. However, only onefragment seeds a cluster. If there is an error in the seeding fragment,the error is repeated in the entire cluster. This error leads tomisidentifying a base and complicating sequencing alignment.

To catch these types of errors, standard barcode sequencing methods usetens to hundreds of copies of the same template, or ten to hundreds ofclusters to create a sample pool for comparison. By drasticallyincreasing the number of copies or clusters, an error can be determined.However, this strategy is expensive and consumes sequencing bandwidth.

SUMMARY

The invention provides methods for increasing base calling accuracy bylinking two fragments originating from the same starting template. Bylinking multiple templates into a single read, information density isincreased and expenses are reduced.

Methods of the present invention have applications in sample preparationand sequencing. In sample preparation methods, the present inventionallows for two identical fragments of a nucleic acid to be joinedtogether. A linking molecule joins the fragments, creating a complex.The complex can include adapters, primers, and binding molecules, inaddition to the identical fragments. Furthermore, in some embodiments,the complex may include multiple identical fragments linked together. Insamples having low target DNA content such as prenatal samples, bylinking two fragments together, fragments can be amplified and sequencedwith increased accuracy.

Methods of the present invention improve base calling when incorporatedinto amplification techniques. In traditional amplification methods,amplicons are created from a single template. If an error exists in thefragment, the error is propagated through the amplification products.Instead of using a single template, both identical templates are used tocreate the amplification products. In the event that there is an errorin either of the two templates, the use of two templates, as opposed toa single template, allows such an error to be identified at thesequencing step.

Methods of the present invention improve amplification on a solidsupport, such as in the Illumina platform (Illumina, Inc. San Diego,CA). In this technique using bridge amplification, clusters of ampliconsare formed. If an error exists in the fragment, the error is repeated inthe cluster. However, with the present invention, linked identicalfragments are contacted to the solid support. The two identicalfragments seed the cluster, resulting in half of the amplicons beingderived from one fragment and the other half are derived from the otherfragment. This technique allows for an error to be readily determined atthe sequencing step.

Methods of the invention improve multiplexing amplification processes.In some embodiments of the present invention, linked fragments can beintroduced into a droplet for amplification. If an error exists ineither fragment, the error is determinable with the raw sequencing data.In some embodiments, the linked fragments can be bound to a microsphereand then with amplification, the fragments seed the microsphere withamplicons. By providing the advantage of forming a plurality ofamplicons using two copies of the same fragment, the present inventionimproves base calling in a variety of applications.

Methods of the invention can be incorporated into multiple sequencingplatforms. For example, in traditional sequencing by synthesis, eachbase is determined sequentially. An error is not determined untilbioinformatics techniques are used to analyze the data. However, thepresent invention allows for two fragments of nucleic acids to be linkedtogether during sequencing methodologies. By analyzing two fragmentssimultaneously, agreement between the two bases indicates accuracy,while disagreement between the two bases would signal an error. With thepresent invention, errors are determinable from the raw sequencing data,without the application of bioinformatics. This technique uses fewercopies or clusters, increases sequencing throughput, and decreasescosts.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E depict the process of forming the linked fragments.

FIG. 2 depicts the linked templates attached to a solid support.

FIG. 3 depicts an example of linked fragments.

FIGS. 4A-4C depict extending and amplification of the linked fragments.

FIGS. 5A-5C depict error determination in the linked fragments.

FIG. 6 depicts adapters and primers

DETAILED DESCRIPTION

The invention generally relates to methods for amplifying and sequencingnucleic acids by joining two copies of fragments. The use of twofragments reduces error rates, increases efficiency in alignment, andreduces sequencing costs.

Nucleic acid generally is acquired from a sample or a subject. Targetmolecules for labeling and/or detection according to the methods of theinvention include, but are not limited to, genetic and proteomicmaterial, such as DNA, genomic DNA, RNA, expressed RNA and/orchromosome(s). Methods of the invention are applicable to DNA from wholecells or to portions of genetic or proteomic material obtained from oneor more cells. Methods of the invention allow for DNA or RNA to beobtained from non-cellular sources, such as viruses. For a subject, thesample may be obtained in any clinically acceptable manner, and thenucleic acid templates are extracted from the sample by methods known inthe art. Generally, nucleic acid can be extracted from a biologicalsample by a variety of techniques such as those described by Maniatis,et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y., pp. 280-281, 1982), the contents of which are incorporated byreference herein in their entirety.

Nucleic acid templates include deoxyribonucleic acid (DNA) and/orribonucleic acid (RNA). Nucleic acid templates can be synthetic orderived from naturally occurring sources. Nucleic acids may be obtainedfrom any source or sample, whether biological, environmental, physicalor synthetic. In one embodiment, nucleic acid templates are isolatedfrom a sample containing a variety of other components, such asproteins, lipids and non-template nucleic acids. Nucleic acid templatescan be obtained from any cellular material, obtained from an animal,plant, bacterium, fungus, or any other cellular organism. Samples foruse in the present invention include viruses, viral particles orpreparations. Nucleic acid may also be acquired from a microorganism,such as a bacteria or fungus, from a sample, such as an environmentalsample.

In the present invention, the target material is any nucleic acid,including DNA, RNA, cDNA, PNA, LNA and others that are contained withina sample. Nucleic acid molecules include deoxyribonucleic acid (DNA)and/or ribonucleic acid (RNA). Nucleic acid molecules can be syntheticor derived from naturally occurring sources. In one embodiment, nucleicacid molecules are isolated from a biological sample containing avariety of other components, such as proteins, lipids and non-templatenucleic acids. Nucleic acid template molecules can be obtained from anycellular material, obtained from an animal, plant, bacterium, fungus, orany other cellular organism. In certain embodiments, the nucleic acidmolecules are obtained from a single cell. Biological samples for use inthe present invention include viral particles or preparations. Nucleicacid molecules can be obtained directly from an organism or from abiological sample obtained from an organism, e.g., from blood, urine,cerebrospinal fluid, seminal fluid, saliva, sputum, stool and tissue.Any tissue or body fluid specimen may be used as a source for nucleicacid for use in the invention. Nucleic acid molecules can also beisolated from cultured cells, such as a primary cell culture or a cellline. The cells or tissues from which template nucleic acids areobtained can be infected with a virus or other intracellular pathogen.In addition, nucleic acids can be obtained from non-cellular ornon-tissue samples, such as viral samples, or environmental samples.

A sample can also be total RNA extracted from a biological specimen, acDNA library, viral, or genomic DNA. In certain embodiments, the nucleicacid molecules are bound as to other target molecules such as proteins,enzymes, substrates, antibodies, binding agents, beads, small molecules,peptides, or any other molecule and serve as a surrogate for quantifyingand/or detecting the target molecule. Generally, nucleic acid can beextracted from a biological sample by a variety of techniques such asthose described by Sambrook and Russell, Molecular Cloning: A LaboratoryManual, Third Edition, Cold Spring Harbor, N.Y. (2001). Nucleic acidmolecules may be single-stranded, double-stranded, or double-strandedwith single-stranded regions (for example, stem- and loop-structures).Proteins or portions of proteins (amino acid polymers) that can bind tohigh affinity binding moieties, such as antibodies or aptamers, aretarget molecules for oligonucleotide labeling, for example, in droplets.

Nucleic acid templates can be obtained directly from an organism or froma biological sample obtained from an organism, e.g., from blood, urine,cerebrospinal fluid, seminal fluid, saliva, sputum, stool and tissue. Ina particular embodiment, nucleic acid is obtained from fresh frozenplasma (FFP). In a particular embodiment, nucleic acid is obtained fromformalin-fixed, paraffin-embedded (FFPE) tissues. Any tissue or bodyfluid specimen may be used as a source for nucleic acid for use in theinvention. Nucleic acid templates can also be isolated from culturedcells, such as a primary cell culture or a cell line. The cells ortissues from which template nucleic acids are obtained can be infectedwith a virus or other intracellular pathogen. A sample can also be totalRNA extracted from a biological specimen, a cDNA library, viral, orgenomic DNA.

A biological sample may be homogenized or fractionated in the presenceof a detergent or surfactant. The concentration of the detergent in thebuffer may be about 0.05% to about 10.0%. The concentration of thedetergent can be up to an amount where the detergent remains soluble inthe solution. In a preferred embodiment, the concentration of thedetergent is between 0.1% to about 2%. The detergent, particularly amild one that is nondenaturing, can act to solubilize the sample.Detergents may be ionic or nonionic. Examples of nonionic detergentsinclude triton, such as the Triton X series (Triton X-100t-Oct-C6H4-(OCH2-CH2)xOH, x=9-10, Triton X-100R, Triton X-114 x=7-8),octyl glucoside, polyoxyethylene(9)dodecyl ether, digitonin, IGEPALCA630 octylphenyl polyethylene glycol, n-octyl-beta-D-glucopyranoside(betaOG), n-dodecyl-beta, Tween 20 polyethylene glycol sorbitanmonolaurate, Tween 80 polyethylene glycol sorbitan monooleate,polidocanol, n-dodecyl beta-D-maltoside (DDM), NP-40 nonylphenylpolyethylene glycol, C12E8 (octaethylene glycol n-dodecyl monoether),hexaethyleneglycol mono-n-tetradecyl ether (C14EO6),octyl-beta-thioglucopyranoside (octyl thioglucoside, OTG), Emulgen, andpolyoxyethylene 10 lauryl ether (C12E10). Examples of ionic detergents(anionic or cationic) include deoxycholate, sodium dodecyl sulfate(SDS), N-lauroylsarcosine, and cetyltrimethylammoniumbromide (CTAB). Azwitterionic reagent may also be used in the purification schemes of thepresent invention, such as Chaps, zwitterion 3-14, and3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulf-onate. It iscontemplated also that urea may be added with or without anotherdetergent or surfactant.

Lysis or homogenization solutions may further contain other agents, suchas reducing agents. Examples of such reducing agents includedithiothreitol (DTT), beta.-mercaptoethanol, DTE, GSH, cysteine,cysteamine, tricarboxyethyl phosphine (TCEP), or salts of sulfurousacid. Once obtained, the nucleic acid is denatured by any method knownin the art to produce single stranded nucleic acid templates and a pairof first and second oligonucleotides is hybridized to the singlestranded nucleic acid template such that the first and secondoligonucleotides flank a target region on the template.

In some embodiments, nucleic acids may be fragmented or broken intosmaller nucleic acid fragments. Nucleic acids, including genomic nucleicacids, can be fragmented using any of a variety of methods, such asmechanical fragmenting, chemical fragmenting, and enzymatic fragmenting.Methods of nucleic acid fragmentation are known in the art and include,but are not limited to, DNase digestion, sonication, mechanicalshearing, and the like (J. Sambrook et al., “Molecular Cloning: ALaboratory Manual”, 1989, 2.sup.nd Ed., Cold Spring Harbour LaboratoryPress: New York, N.Y.; P. Tijssen, “Hybridization with Nucleic AcidProbes—Laboratory Techniques in Biochemistry and Molecular Biology(Parts I and II)”, 1993, Elsevier; C. P. Ordahl et al., Nucleic AcidsRes., 1976, 3: 2985-2999; P. J. Oefner et al., Nucleic Acids Res., 1996,24: 3879-3889; Y. R. Thorstenson et al., Genome Res., 1998, 8: 848-855).U.S. Patent Publication 2005/0112590 provides a general overview ofvarious methods of fragmenting known in the art.

Genomic nucleic acids can be fragmented into uniform fragments orrandomly fragmented. In certain aspects, nucleic acids are fragmented toform fragments having a fragment length of about 5 kilobases or 100kilobases. In a preferred embodiment, the genomic nucleic acid fragmentscan range from 1 kilobases to 20 kilobases. Preferred fragments can varyin size and have an average fragment length of about 10 kilobases.However, desired fragment length and ranges of fragment lengths can beadjusted depending on the type of nucleic acid targets one seeks tocapture. The particular method of fragmenting is selected to achieve thedesired fragment length. A few non-limiting examples are provided below.

Chemical fragmentation of genomic nucleic acids can be achieved using anumber of different methods. For example, hydrolysis reactions includingbase and acid hydrolysis are common techniques used to fragment nucleicacid. Hydrolysis is facilitated by temperature increases, depending uponthe desired extent of hydrolysis. Fragmentation can be accomplished byaltering temperature and pH as described below. The benefit of pH-basedhydrolysis for shearing is that it can result in single-strandedproducts. Additionally, temperature can be used with certain buffersystems (e.g. Tris) to temporarily shift the pH up or down from neutralto accomplish the hydrolysis, then back to neutral for long-term storageetc. Both pH and temperature can be modulated to affect differingamounts of shearing (and therefore varying length distributions).

Other methods of hydrolytic fragmenting of nucleic acids includealkaline hydrolysis, formalin fixation, hydrolysis by metal complexes(e.g., porphyrins), and/or hydrolysis by hydroxyl radicals. RNA shearsunder alkaline conditions, see, e.g. Nordhoff et al., Nucl. Acid. Res.,21 (15):3347-57 (2003), whereas DNA can be sheared in the presence ofstrong acids.

An exemplary acid/base hydrolysis protocol for producing genomic nucleicacid fragments is described in Sargent et al. (1988) Methods Enzymol.,152:432. Briefly, 1 g of purified DNA is dissolved in 50 mL 0.1 N NaOH.1.5 mL concentrated HCl is added and the solution is mixed quickly. DNAwill precipitate immediately, and should not be stirred for more than afew seconds to prevent formation of a large aggregate. The sample isincubated at room temperature for 20 minutes to partially depurinate theDNA. Subsequently, 2 mL 10 N NaOH (OH— concentration to 0.1 N) is added,and the sample is stirred until the DNA redissolves completely. Thesample is then incubated at 65 degrees C. for 30 minutes in order tohydrolyze the DNA. Resulting fragments typically range from about250-1000 nucleotides but can vary lower or higher depending on theconditions of hydrolysis.

In one embodiment, after genomic nucleic acid has been purified, it isresuspended in a Tris-based buffer at a pH between 7.5 and 8.0, such asQiagen's DNA hydrating solution. The resuspended genomic nucleic acid isthen heated to 65 C and incubated overnight. Heating shifts the pH ofthe buffer into the low- to mid-6 range, which leads to acid hydrolysis.Over time, the acid hydrolysis causes the genomic nucleic acid tofragment into single-stranded and/or double-stranded products.

Chemical cleavage can also be specific. For example, selected nucleicacid molecules can be cleaved via alkylation, particularlyphosphorothioate-modified nucleic acid molecules (see, e.g., K. A.Browne, “Metal ion-catalyzed nucleic Acid alkylation and fragmentation,”J. Am. Chem. Soc. 124(27):7950-7962 (2002)). Alkylation at thephosphorothioate modification renders the nucleic acid moleculesusceptible to cleavage at the modification site. See I. G. Gut and S.Beck, “A procedure for selective DNA alkylation and detection by massspectrometry,” Nucl. Acids Res. 23(8):1367-1373 (1995).

Methods of the invention also contemplate chemically shearing nucleicacids using the technique disclosed in Maxam-Gilbert Sequencing Method(Chemical or Cleavage Method), Proc. Natl. Acad. Sci. USA. 74:560-564.In that protocol, the genomic nucleic acid can be chemically cleaved byexposure to chemicals designed to fragment the nucleic acid at specificbases, such as preferential cleaving at guanine, at adenine, at cytosineand thymine, and at cytosine alone.

Mechanical shearing of nucleic acids into fragments can occur using anymethod known in the art. For example, fragmenting nucleic acids can beaccomplished by hydroshearing, trituration through a needle, andsonication. See, for example, Quail, et al. (November 2010) DNA:Mechanical Breakage. In: eLS. John Wiley & Sons, Chichester.doi:10.1002/9780470015902.a0005 333.pub2.

The nucleic acid can also be sheared via nebulization, see (Roe, B A,Crabtree. J S and Khan, A S 1996); Sambrook & Russell, Cold Spring HarbProtoc 2006. Nebulizing involves collecting fragmented DNA from a mistcreated by forcing a nucleic acid solution through a small hole in anebulizer. The size of the fragments obtained by nebulization isdetermined chiefly by the speed at which the DNA solution passes throughthe hole, altering the pressure of the gas blowing through thenebulizer, the viscosity of the solution, and the temperature. Theresulting DNA fragments are distributed over a narrow range of sizes(700-1330 bp). Shearing of nucleic acids can be accomplished by passingobtained nucleic acids through the narrow capillary or orifice (Oefneret al., Nucleic Acids Res. 1996; Thorstenson et al., Genome Res. 1995).This technique is based on point-sink hydrodynamics that result when anucleic acid sample is forced through a small hole by a syringe pump.

In HydroShearing (Genomic Solutions, Ann Arbor, Mich., USA), DNA insolution is passed through a tube with an abrupt contraction. As itapproaches the contraction, the fluid accelerates to maintain thevolumetric flow rate through the smaller area of the contraction. Duringthis acceleration, drag forces stretch the DNA until it snaps. The DNAfragments until the pieces are too short for the shearing forces tobreak the chemical bonds. The flow rate of the fluid and the size of thecontraction determine the final DNA fragment sizes.

Sonication is also used to fragment nucleic acids by subjecting thenucleic acid to brief periods of sonication, i.e. ultrasound energy. Amethod of shearing nucleic acids into fragments by sonification isdescribed in U.S. Patent Publication 2009/0233814. In the method, apurified nucleic acid is obtained placed in a suspension havingparticles disposed within. The suspension of the sample and theparticles are then sonicated into nucleic acid fragments.

An acoustic-based system that can be used to fragment DNA is describedin U.S. Pat. Nos. 6,719,449, and 6,948,843 manufactured by Covaris Inc.U.S. Pat. No. 6,235,501 describes a mechanical focusing acousticsonication method of producing high molecular weight DNA fragments byapplication of rapidly oscillating reciprocal mechanical energy in thepresence of a liquid medium in a closed container, which may be used tomechanically fragment the DNA.

Another method of shearing nucleic acids into fragments uses ultrasoundenergy to produce gaseous cavitation in liquids, such as shearing withDiagonnode's BioRuptor (electrical shearing device, commerciallyavailable by Diagenode, Inc.). Cavitation is the formation of smallbubbles of dissolved gases or vapors due to the alteration of pressurein liquids. These bubbles are capable of resonance vibration and producevigorous eddying or microstreaming. The resulting mechanical stress canlead to shearing the nucleic acid in to fragments.

Enzymatic fragmenting, also known as enzymatic cleavage, cuts nucleicacids into fragments using enzymes, such as endonucleases, exonucleases,ribozymes, and DNAzymes. Such enzymes are widely known and are availablecommercially, see Sambrook, J. Molecular Cloning: A Laboratory Manual,3rd (2001) and Roberts R J (January 1980). “Restriction and modificationenzymes and their recognition sequences,” Nucleic Acids Res. 8 (1):r63-r80. Varying enzymatic fragmenting techniques are well-known in theart, and such techniques are frequently used to fragment a nucleic acidfor sequencing, for example, Alazard et al, 2002; Bentzley et al, 1998;Bentzley et al, 1996; Faulstich et al, 1997; Glover et al, 1995;Kirpekar et al, 1994; Owens et al, 1998; Pieles et al, 1993; Schuette etal, 1995; Smirnov et al, 1996; Wu & Aboleneen, 2001; Wu et al, 1998a.

The most common enzymes used to fragment nucleic acids areendonucleases. The endonucleases can be specific for either adouble-stranded or a single stranded nucleic acid molecule. The cleavageof the nucleic acid molecule can occur randomly within the nucleic acidmolecule or can cleave at specific sequences of the nucleic acidmolecule. Specific fragmentation of the nucleic acid molecule can beaccomplished using one or more enzymes in sequential reactions orcontemporaneously.

Restriction endonucleases recognize specific sequences withindouble-stranded nucleic acids and generally cleave both strands eitherwithin or close to the recognition site in order to fragment the nucleicacid. Naturally occurring restriction endonucleases are categorized intofour groups (Types I, II III, and IV) based on their composition andenzyme cofactor requirements, the nature of their target sequence, andthe position of their DNA cleavage site relative to the target sequence.Bickle T A, Krüger D H (June 1993), “Biology of DNA restriction,”Microbiol. Rev. 57 (2): 434-50; Boyer H W (1971). “DNA restriction andmodification mechanisms in bacteria”. Annu. Rev. Microbiol. 25: 153-76;Yuan R (1981). “Structure and mechanism of multifunctional restrictionendonucleases”. Annu. Rev. Biochem. 50: 285-319. All types of enzymesrecognize specific short DNA sequences and carry out the endonucleolyticcleavage of DNA to give specific fragments with terminal 5′-phosphates.The enzymes differ in their recognition sequence, subunit composition,cleavage position, and cofactor requirements. Williams R J (2003).“Restriction endonucleases: classification, properties, andapplications”. Mol. Biotechnol. 23 (3): 225-43.

Where restriction endonucleases recognize specific sequencings indouble-stranded nucleic acids and generally cleave both strands, nickingendonucleases are capable of cleaving only one of the strands of thenucleic acid into a fragment. Nicking enzymes used to fragment nucleicacids can be naturally occurring or genetically engineered fromrestriction enzymes. See Chan et al., Nucl. Acids Res. (2011) 39 (1):1-18.

In some embodiments, DNA is sheared in biological processes within anorganism, or a biological medium. Such DNA, or cell-free DNA, circulatesfreely in the blood stream. For example, cell-free fetal DNA (cffDNA) isfetal DNA that circulates freely in the maternal blood stream. Someembodiments use fragmented or sheared DNA, however, the DNA is obtainedin fragmented form.

In preferred embodiments of the present invention, the nucleic acidfragments are joined together in a complex, for example, see FIG. 3 .Any linking molecule may be used to join the molecules. The linker usedin the present invention may be synthesized or obtained commerciallyfrom various companies, for example, Integrated DNA Technologies, Inc.,Gene Link, Inc., and TriLink Biotechnologies, Inc. The linker may be anymolecule to join two primers or two nucleic acid fragments. The linkingmolecule may also join multiple fragments together. Any number offragments may be incorporated to the complex.

The linking molecule may also serve to separate the nucleic acidfragments. In preferred embodiments, the fragments are oriented toprevent binding there between. With the linker creating spatialseparation and orientation of the fragments controlled, collapsing orbinding between the fragments can be avoided and prevented.

In some embodiments the linkers may be polyethylene glycol (PEG) or amodified PEG. A modified PEG, such as DBCO-PEG₄, or PEG-11 may be usedto join the two adapters or nucleic acids. In another example,N-hydroxysuccinimide (NETS) modified PEG is used to join the twoadapters. See Schlingman, et al., Colloids and Surfaces B: Biointerfaces83 (2011) 91-95. Any oligonucleotide or other molecule may be used tojoin adapters or nucleic acids.

In some embodiments, aptamers are used to bind two adapters or nucleicacids. Aptamers can be designed to bind to various molecular targets,such as primers or nucleic acids. Aptamers may be designed or selectedby the SELEX (systematic evolution of ligands by exponential enrichment)method. Aptamers are nucleic acid macromolecules that specifically bindto target molecules. Like all nucleic acids, a particular nucleic acidligand, i.e., an aptamer, may be described by a linear sequence ofnucleotides (A, U, T, C and G), typically 15-40 nucleotides long. Insome preferred embodiments, the aptamers may include inverted bases ormodified bases. In some embodiments, aptamers or modified aptamers,include at least one inverted base or modified base.

It should be appreciated that the linker may be composed of invertedbases, or comprise at least one inverted base. Inverted bases ormodified bases may be acquired through any commercial entity. Invertedbases or modified bases are developed and commercially available.Inverted bases or modified bases may be incorporated into othermolecules. For example, 2-Aminopurine can be substituted in anoligonucleotide. 2-Aminopurine is a fluorescent base that is useful as aprobe for monitoring the structure and dynamics of DNA.2,6-Diaminopurine (2-Amino-dA) is a modified base can form threehydrogen bonds when base-paired with dT and can increase the Tm of shortoligos. 5-Bromo-deoxyuridine is a photoreactive halogenated base thatcan be incorporated into oligonucleotides to crosslink them to DNA, RNAor proteins with exposure to UV light. Other examples of inverted basesor modified bases include deoxyUridine (dU), inverted dT,dideoxycytidine (ddC), 5-methyl deoxyCytidine, or 2′-deoxyInosine (dI).It should be appreciated that any inverted or modified based can be usedin linking template nucleic acids.

In preferred embodiments, the linker comprises a molecule for joiningtwo primers or two nucleic acid fragments. The linker may be a singlemolecule, or a plurality of molecules. The linker may comprise a fewinverted bases or modified bases, or entirely inverted bases or modifiedbases. The linker may comprise a both Watson-Crick bases and inverted ormodified bases.

It should be appreciated that any spacer molecule or linking moleculemay be used in the present invention. In some embodiments, the linker orspacer molecule may be a lipid or an oligosaccharide, or anoligosaccharide and a lipid. See U.S. Pat. No. 5,122,450. In thisexample, the molecule is preferably a lipid molecule and, morepreferably, a glyceride or phosphatide which possesses at least twohydrophobic polyalkylene chains.

The linker may be composed of any number of adapters, primers, andcopies of fragments. A linker may include two identical arms, where eacharm is composed of binding molecules, amplification primers, sequencingprimers, adapters, and fragments. A linker may link together any numberof arms, such as three or four arms. It should be appreciated that insome aspects of the invention, nucleic acid templates are linked by aspacer molecule. The linker in the present invention may be any moleculeor method to join two fragments or primers. In some embodiments,polyethylene glycol or a modified PEG such as DBCO-PEG₄ or PEG-11 isused. In some embodiments the linker is a lipid or a hydrocarbon. Insome embodiments a protein may join the adapters or the nucleic acids.In some embodiments, an oligosaccharide links the primers or nucleicacids. In some embodiments, aptamers link the primers or nucleic acids.When the fragments are linked, the copies are oriented to be in phase soto prevent binding there between.

In certain embodiments, a linker may be an antibody. The antibody may bea monomer, a dimer or a pentamer. It should be appreciated that anyantibody for joining two primers or nucleic acids may be used. Forexample, it is known in the art that nucleoside can be made immunogenicby coupling to proteins. See Void, BS (1979), Nucl Acids Res 7, 193-204.In addition, antibodies may be prepared to bind to modified nucleicacids. See Biochemical Education, Vol. 12, Issue 3.

The linker may stay attached to the complex during amplification. Insome embodiments, the linker is removed prior to amplification. In someembodiments, a linker is attached to a binding molecule, and the bindingmolecule is then attached to an amplification primer. When the linker isremoved, the binding molecule or binding primer is exposed. The exposedbinding molecule also attaches to a solid support and an arch is formed.The linker may be removed by any known method in the art, includingwashing with a solvent, applying heat, altering pH, washing with adetergent or surfactant, etc.

Methods of the invention provide for nucleic acids to be linked togetherwith a linker molecule. In samples with low genetic material, nucleicacids can be linked together in order to ensure identical fragmentsamplified simultaneously or sequentially. Samples such as prenatalsamples have low genetic content and amplifying identical fragmentsincreases the detectable content. This method reduces the signal tonoise ratio, improving the detection of the target sequence.

Methods of the invention utilize amplification to amplify a targetnucleic acid, such as a fragment, to a detectable level. It should beappreciated that any known amplification technique can be used in thepresent invention. Further, the amplified segments created by anamplification process may be themselves, efficient templates forsubsequent amplifications.

Amplification refers to production of additional copies of a nucleicacid sequence and is generally carried out using polymerase chainreaction or other technologies well known in the art (e.g., Dieffenbachand Dveksler, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y. [1995]). The amplification reaction may be anyamplification reaction known in the art that amplifies nucleic acidmolecules, such as polymerase chain reaction, nested polymerase chainreaction, ligase chain reaction (Barany F. (1991) PNAS 88:189-193;Barany F. (1991) PCR Methods and Applications 1:5-16), ligase detectionreaction (Barany F. (1991) PNAS 88:189-193), transcription basedamplification system, nucleic acid sequence-based amplification, rollingcircle amplification, and hyper-branched rolling circle amplification.

In some embodiments, multiple displacement amplification (MDA), anon-PCR based DNA amplification technique, rapidly amplifies minuteamounts of DNA samples for genomic analysis. The reaction starts byannealing random hexamer primers to the template: DNA synthesis iscarried out by a high fidelity enzyme at a constant temperature.However, it should be appreciated that any amplification method may beused with the current invention.

In certain embodiments of the invention, the amplification reaction isthe polymerase chain reaction. Polymerase chain reaction (PCR) refers tomethods by K. B. Mullis (U.S. Pat. Nos. 4,683,195 and 4,683,202, herebyincorporated by reference) for increasing concentration of a segment ofa target sequence in a mixture of genomic DNA without cloning orpurification. The process for amplifying the target sequence includesintroducing an excess of oligonucleotide primers to a DNA mixturecontaining a desired target sequence, followed by a precise sequence ofthermal cycling in the presence of a DNA polymerase. The primers arecomplementary to their respective strands of the double stranded targetsequence.

In some aspects of the invention, PCR primers are joined by a linkermolecule and through the PCR process, identical copies of a fragment islinked to the primers. In other embodiments, adapters are added to theprimers or copies of the fragments. The resulting complex includes,generally, two identical copies of a fragment directly or indirectlyjoined by a linking molecule. It should be appreciated that although thecopies of the same fragment, due to amplification errors, one or bothcopies may include an error. However, there is a low probability thateach fragment will have an error at the exact same base. Disagreementbetween the two fragments at a base would indicate an error. The basecould then be identified as an unknown, just from the raw sequencingdata.

Primers can be prepared by a variety of methods including but notlimited to cloning of appropriate sequences and direct chemicalsynthesis using methods well known in the art (Narang et al., MethodsEnzymol., 68:90 (1979); Brown et al., Methods Enzymol., 68:109 (1979)).Primers can also be obtained from commercial sources such as OperonTechnologies, Amersham Pharmacia Biotech, Sigma, and Life Technologies.The primers can have an identical melting temperature. The lengths ofthe primers can be extended or shortened at the 5′ end or the 3′ end toproduce primers with desired melting temperatures. Also, the annealingposition of each primer pair can be designed such that the sequence and,length of the primer pairs yield the desired melting temperature. Thesimplest equation for determining the melting temperature of primerssmaller than 25 base pairs is the Wallace Rule (Td=2(A+T)+4(G+C)).Computer programs can also be used to design primers, including but notlimited to Array Designer Software (Arrayit Inc.), Oligonucleotide ProbeSequence Design Software for Genetic Analysis (Olympus Optical Co.),NetPrimer, and DNAs is from Hitachi Software Engineering. The TM(melting or annealing temperature) of each primer is calculated usingsoftware programs such as Oligo Design, available from Invitrogen Corp.

In some embodiments, to effect amplification, a mixture is denatured andthe primers then annealed to their complementary sequences within thetarget molecule. Following annealing, the primers are extended with apolymerase so as to form a new pair of complementary strands. The stepsof denaturation, primer annealing and polymerase extension can berepeated many times (i.e., denaturation, annealing and extensionconstitute one cycle; there can be numerous cycles) to obtain a highconcentration of an amplified segment of a desired target sequence. Thelength of the amplified segment of the desired target sequence isdetermined by relative positions of the primers with respect to eachother, and therefore, this length is a controllable parameter.

In some embodiments, to create complexes of the invention, primers arelinked by a linking molecule or a spacer molecule to create two linkedcopies of the fragment. In other embodiments, two fragments are linkedtogether following at least one PCR step. It should be appreciated thatPCR can be applied to fragments before or after the fragments are joinedvia a linking molecule. In some embodiments, when the fragments arejoined, PCR can be implemented on the joined fragments. In someembodiments, the linked copies undergo amplification. The amplificationstep includes linked primers. The result is that after a cycle of PCR,linked complexes comprising copies of the fragments are produced.

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level that can be detected by severaldifferent methodologies (e.g., staining, hybridization with a labeledprobe; incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of 32P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment). Inaddition to genomic DNA, any oligonucleotide sequence can be amplifiedwith the appropriate set of primer molecules. In particular, theamplified segments created by the PCR process itself are, themselves,efficient templates for subsequent PCR amplifications. Amplified targetsequences can be used to obtain segments of DNA (e.g., genes) forinsertion into recombinant vectors.

Other amplification methods and strategies can also be utilized in thepresent invention. For example, another approach would be to combine PCRand the ligase chain reaction (LCR). Since PCR amplifies faster than LCRand requires fewer copies of target DNA to initiate, PCR can be used asfirst step followed by LCR. The amplified product could then be used ina LCR or ligase detection reaction (LDR) in an allele-specific mannerthat would indicate if a mutation was present. Another approach is touse LCR or LDR for both amplification and allele-specificdiscrimination. The later reaction is advantageous in that it results inlinear amplification. Thus the amount of amplified product is areflection of the amount of target DNA in the original specimen andtherefore permits quantitation.

LCR utilizes pairs of adjacent oligonucleotides which are complementaryto the entire length of the target sequence (Barany F. (1991) PNAS88:189-193; Barany F. (1991) PCR Methods and Applications 1:5-16). Ifthe target sequence is perfectly complementary to the primers at thejunction of these sequences, a DNA ligase will link the adjacent 3′ and5′ terminal nucleotides forming a combined sequence. If a thermostableDNA ligase is used with thermal cycling, the combined sequence will besequentially amplified. A single base mismatch at the junction of theoligonucleotides will preclude ligation and amplification. Thus, theprocess is allele-specific. Another set of oligonucleotides with 3′nucleotides specific for the mutant would be used in another reaction toidentify the mutant allele. A series of standard conditions could beused to detect all possible mutations at any known site. LCR typicallyutilizes both strands of genomic DNA as targets for oligonucleotidehybridization with four primers, and the product is increasedexponentially by repeated thermal cycling.

Amplification or sequencing adapters or barcodes, or a combinationthereof, may be attached to the fragmented nucleic acid. Such moleculesmay be commercially obtained, such as from Integrated DNA Technologies(Coralville, IA). In certain embodiments, such sequences are attached tothe template nucleic acid molecule with an enzyme such as a ligase.Suitable ligases include T4 DNA ligase and T4 RNA ligase, availablecommercially from New England Biolabs (Ipswich, MA). The ligation may beblunt ended or via use of complementary overhanging ends.

In certain embodiments, following fragmentation, the ends of thefragments may be repaired, trimmed (e.g. using an exonuclease), orfilled (e.g., using a polymerase and dNTPs) to form blunt ends. In someembodiments, end repair is performed to generate blunt end 5′phosphorylated nucleic acid ends using commercial kits, such as thoseavailable from Epicentre Biotechnologies (Madison, WI). Upon generatingblunt ends, the ends may be treated with a polymerase and dATP to form atemplate independent addition to the 3′-end and the 5′-end of thefragments, thus producing a single A overhanging. This single A canguide ligation of fragments with a single T overhanging from the 5′-endin a method referred to as T-A cloning. Alternatively, because thepossible combination of overhangs left by the restriction enzymes areknown after a restriction digestion, the ends may be left as-is, i.e.,ragged ends. In certain embodiments double stranded oligonucleotideswith complementary overhanging ends are used.

In certain embodiments, one or more bar code is attached to each, any,or all of the fragments. A bar code sequence generally includes certainfeatures that make the sequence useful in sequencing reactions. The barcode sequences are designed such that each sequence is correlated to aparticular portion of nucleic acid, allowing sequence reads to becorrelated back to the portion from which they came. Methods ofdesigning sets of bar code sequences is shown for example in U.S. Pat.No. 6,235,475, the contents of which are incorporated by referenceherein in their entirety. In certain embodiments, the bar code sequencesare attached to the template nucleic acid molecule, e.g., with anenzyme. The enzyme may be a ligase or a polymerase, as discussed above.Attaching bar code sequences to nucleic acid templates is shown in U.S.Pub. 2008/0081330 and U.S. Pub. 2011/0301042, the content of each ofwhich is incorporated by reference herein in its entirety. Methods fordesigning sets of bar code sequences and other methods for attaching barcode sequences are shown in U.S. Pat. Nos. 7,537,897; 6,138,077;6,352,828; 5,636,400; 6,172,214; and 5,863,722, the content of each ofwhich is incorporated by reference herein in its entirety. After anyprocessing steps (e.g., obtaining, isolating, fragmenting,amplification, or barcoding), nucleic acid can be sequenced.

Exemplary methods for designing sets of barcode sequences and othermethods for attaching barcode sequences are shown in U.S. Pat. Nos.6,138,077; 6,352,828; 5,636,400; 6,172,214; 6,235,475; 7,393,665;7,544,473; 5,846,719; 5,695,934; 5,604,097; 6,150,516; RE39,793;7,537,897; 6,172,218; and 5,863,722, the content of each of which isincorporated by reference herein in its entirety.

The barcode sequence generally includes certain features that make thesequence useful in sequencing reactions. For example the barcodesequences can be designed to have minimal or no homopolymer regions,i.e., 2 or more of the same base in a row such as AA or CCC, within thebarcode sequence. The barcode sequences can also be designed so thatthey do not overlap the target region to be sequence or contain asequence that is identical to the target.

The first and second barcode sequences are designed such that each pairof sequences is correlated to a particular sample, allowing samples tobe distinguished and validated. Methods of designing sets of barcodesequences is shown for example in Brenner et al. (U.S. Pat. No.6,235,475), the contents of which are incorporated by reference hereinin their entirety. In certain embodiments, the barcode sequences rangefrom about 2 nucleotides to about 50; and preferably from about 4 toabout 20 nucleotides. Since the barcode sequence is sequenced along withthe template nucleic acid or may be sequenced in a separate read, theoligonucleotide length should be of minimal length so as to permit thelongest read from the template nucleic acid attached. Generally, thebarcode sequences are spaced from the template nucleic acid molecule byat least one base.

Methods of the invention involve attaching the barcode sequences to thetemplate nucleic acids. Template nucleic acids are able to be fragmentedor sheared to desired length, e.g. generally from 100 to 500 bases orlonger, using a variety of mechanical, chemical and/or enzymaticmethods. DNA may be randomly sheared via sonication, exposed to a DNaseor one or more restriction enzymes, a transposase, or nicking enzyme.RNA may be fragmented by brief exposure to an RNase, heat plusmagnesium, or by shearing. The RNA may be converted to cDNA before orafter fragmentation.

Barcode sequence is integrated with template using methods known in theart. Barcode sequence is integrated with template using, for example, aligase, a polymerase, Topo cloning (e.g., Invitrogen's topoisomerasevector cloning system using a topoisomerase enzyme), or chemicalligation or conjugation. The ligase may be any enzyme capable ofligating an oligonucleotide (RNA or DNA) to the template nucleic acidmolecule. Suitable ligases include T4 DNA ligase and T4 RNA ligase (suchligases are available commercially, from New England Biolabs). Methodsfor using ligases are well known in the art. The polymerase may be anyenzyme capable of adding nucleotides to the 3′ and the 5′ terminus oftemplate nucleic acid molecules. Barcode sequence can be incorporatedvia a PCR reaction as part of the PCR primer. Regardless of theincorporation of molecular barcodes or the location of the barcodes inthe event that they are incorporated, sequencing adaptors can beattached to the nucleic acid product in a bi-directional way such thatin the same sequencing run there will be sequencing reads from both the5′ and 3′ end of the target sequence. In some cases it is advantage touse the location of the barcode on the 5′ or 3′ end of the targetsequence to indicate the direction of the read. It is well known to oneskilled in the art how to attach the sequencing adaptors usingtechniques such as PCR or ligation.

FIG. 6 shows examples of possible configurations of adapter and primers.As shown at 602, a P7 primer is attached to a Read2 primer site, whichis attached to a complimentary region. At 603, a linked PCR primingregion is attached to a unique molecular identifier. As shown at 604, aP5 primer is attached to an index read primer site, and a seedingcontrol site.

In some embodiments, multiple copies of a fragment are joined together.It should be appreciated that any number of fragments can be joinedtogether, whether 2, 3, 4, etc. The joined copies may be referred to asa unit. Several units may then be joined together with a linkingmolecule. It should be appreciated that any number of units may bejoined by a linking molecule. This increases the information densitywithin a complex. When the complex is attached to a solid support, thecomplex is amplified. The amplification products may be attached to thesolid support. By joining multiple copies of the fragment to the complexand then amplifying the complexes, information density on a solidsupport increases.

In certain embodiments, the nucleic acids may be amplified by two joinedprimers. As shown in FIG. 1A, a linker 103 comprises two short primers105 with concentration driven Tm. The linker 103 or the primers 105 maybe also attached to universal adapters (not shown). During linear PCR,two copies of the genomic template 107 are prepared. As shown in FIG.1B, the complex 109 comprises the linker 103, the primers 105 andidentical copies of the nucleic acid template 107. As shown in FIG. 1C,a second linear PCR step using a different linker 116 and adapters 118is used to create the opposite senses 114 to nucleic acid templates 107.Complexes 109 and 119 undergo additional steps of amplification, such asuniversal PCR, to create multiple amplicons of both senses (the senseand anti-sense). See for FIGS. 1D and 1E.

An example complex is shown in FIG. 3 . As shown in FIG. 3 , a complex301 contains a linker 301 attached to two primers 303. Complex alsocomprises sequence read primers 305 and adapters 307 to link to thetarget nucleic acid 309. The complex also comprises complimentaryadapters 311 and primers 313. In a preferred embodiment, primers 313 areP7 primers and primers 303 are P5 primers. It should be appreciated thatany combination, orientation or configuration of the adapters, primers,and target nucleic acids can be organized. It should also be appreciatedthat the complexes may include bar codes. FIG. 3 is to be an example andnot a limiting embodiment.

Complexes of the invention, which comprise a linking molecule, identicalfragments of the nucleic acid, and optionally, adapters and primers maybe incorporated into partitions, such as emulsions, droplets, wellplates, etc. The droplets may be aqueous droplets surrounded by animmiscible carrier fluid. Methods of forming such droplets are shown forexample in Link et al. (U.S. patent application numbers 2008/0014589,2008/0003142, and 2010/0137163), Stone et al. (U.S. Pat. No. 7,708,949and U.S. patent application number 2010/0172803), and Anderson et al.(U.S. Pat. No. 7,041,481 and which reissued as RE41,780). Complexes ofthe invention may be attached to various solid supports such asmicrobeads, beads, channel walls, microchips, etc.

Sequencing the joined identical fragments may be by any method known inthe art. The present invention has applications in various sequencingplatforms, including the genome sequencers from Roche/454 Life Sciences(Margulies et al. (2005) Nature, 437:376-380; U.S. Pat. Nos. 6,274,320;6,258,568; 6,210,891), the SOLiD system from Life Technologies AppliedBiosystems (Grand Island, NY), the HELISCOPE system from HelicosBiosciences (Cambridge, MA) (see, e.g., U.S. Pub. 2007/0070349), and theIon sequencers from Life Technologies Ion Torrent, Ion Torrent Systems,Inc. (Guilford, CT).

In preferred embodiments, sequencing is by methods where each base isdetermine sequentially. DNA sequencing techniques include classicdideoxy sequencing reactions (Sanger method) using labeled terminatorsor primers and gel separation in slab or capillary, sequencing bysynthesis using reversibly terminated labeled nucleotides,pyrosequencing, 454 sequencing, allele specific hybridization to alibrary of labeled oligonucleotide probes, sequencing by synthesis usingallele specific hybridization to a library of labeled clones that isfollowed by ligation, real time monitoring of the incorporation oflabeled nucleotides during a polymerization step, polony sequencing, andSOLiD sequencing. Sequencing of separated molecules has more recentlybeen demonstrated by sequential or single extension reactions usingpolymerases or ligases as well as by single or sequential differentialhybridizations with libraries of probes.

It should be appreciated that the linker may also be attached toadapters, primers, or binding molecules. The linker can be attached tothese species in any orientation or arrangement. The linking moleculemay be directly attached to an adapter or primer and indirectly linkedto the nucleic acid fragments. In some aspects of the invention, thelinking molecule is removed before or after amplification. In someembodiments, the linking molecule remains on the complex. In someembodiments, the linking molecule is removed prior to sequencing, wherein other embodiments the linking molecule remains on the complex duringsequencing.

A sequencing technique that can be used in the methods of the providedinvention includes, for example, Helicos True Single Molecule Sequencing(tSMS) (Harris T. D. et al. (2008) Science 320:106-109). In the tSMStechnique, a DNA sample is cleaved into strands of approximately 100 to200 nucleotides, and a polyA sequence is added to the 3′ end of each DNAstrand. Each strand is labeled by the addition of a fluorescentlylabeled adenosine nucleotide. The DNA strands are then hybridized to aflow cell, which contains millions of oligo-T capture sites that areimmobilized to the flow cell surface. The templates can be at a densityof about 100 million templates/cm². The flow cell is then loaded into aninstrument, e.g., HeliScope sequencer, and a laser illuminates thesurface of the flow cell, revealing the position of each template. A CCDcamera can map the position of the templates on the flow cell surface.The template fluorescent label is then cleaved and washed away. Thesequencing reaction begins by introducing a DNA polymerase and afluorescently labeled nucleotide. The oligo-T nucleic acid serves as aprimer. The polymerase incorporates the labeled nucleotides to theprimer in a template directed manner. The polymerase and unincorporatednucleotides are removed. The templates that have directed incorporationof the fluorescently labeled nucleotide are detected by imaging the flowcell surface. After imaging, a cleavage step removes the fluorescentlabel, and the process is repeated with other fluorescently labelednucleotides until the desired read length is achieved. Sequenceinformation is collected with each nucleotide addition step. With thepresent invention, the linked fragments can be identified in tandem.Further description of tSMS is shown for example in Lapidus et al. (U.S.Pat. No. 7,169,560), Lapidus et al. (U.S. patent application number2009/0191565), Quake et al. (U.S. Pat. No. 6,818,395), Harris (U.S. Pat.No. 7,282,337), Quake et al. (U.S. patent application number2002/0164629), and Braslaysky, et al., PNAS (USA), 100: 3960-3964(2003), the contents of each of these references is incorporated byreference herein in its entirety.

Another example of a DNA sequencing technique that can be used in themethods of the provided invention is 454 sequencing (Roche) (Margulies,M et al. 2005, Nature, 437, 376-380). 454 sequencing involves two steps.In the first step, DNA is sheared into fragments of approximately300-800 base pairs, and the fragments are blunt ended. Oligonucleotideadaptors are then ligated to the ends of the fragments. The adaptorsserve as primers for amplification and sequencing of the fragments. Thefragments can be attached to DNA capture beads, e.g.,streptavidin-coated beads using, e.g., Adaptor B, which contains5′-biotin tag. Using the methods of the present invention, joinedfragments as described above are captured on the beads. The joinedfragments attached to the beads are PCR amplified within droplets of anoil-water emulsion. The result is multiple copies of clonally amplifiedDNA fragments on each bead. In the second step, the beads are capturedin wells (pico-liter sized). Pyrosequencing is performed on each DNAfragment in parallel. Addition of one or more nucleotides generates alight signal that is recorded by a CCD camera in a sequencinginstrument. The signal strength is proportional to the number ofnucleotides incorporated. Pyrosequencing makes use of pyrophosphate(PPi) which is released upon nucleotide addition. PPi is converted toATP by ATP sulfurylase in the presence of adenosine 5′ phosphosulfate.Luciferase uses ATP to convert luciferin to oxyluciferin, and thisreaction generates light that is detected and analyzed.

Another example of a DNA sequencing technique that can be used in themethods of the provided invention is Ion Torrent sequencing (U.S. patentapplication numbers 2009/0026082, 2009/0127589, 2010/0035252,2010/0137143, 2010/0188073, 2010/0197507, 2010/0282617, 2010/0300559),2010/0300895, 2010/0301398, and 2010/0304982), the content of each ofwhich is incorporated by reference herein in its entirety. In IonTorrent sequencing, DNA is sheared into fragments of approximately300-800 base pairs, and the fragments are blunt ended. Oligonucleotideadaptors are then ligated to the ends of the fragments. The adaptorsserve as primers for amplification and sequencing of the fragments. Thefragments can be attached to a surface and is attached at a resolutionsuch that the fragments are individually resolvable. Using the methodsof the present invention, the joined fragments are attached to thesurface. Addition of one or more nucleotides releases a proton (H+),which signal detected and recorded in a sequencing instrument. Thesignal strength is proportional to the number of nucleotidesincorporated.

The invention also encompasses methods of sequencing amplified nucleicacids generated by solid-phase amplification. Thus, the inventionprovides a method of nucleic acid sequencing comprising amplifying apool of nucleic acid templates using solid-phase amplification andcarrying out a nucleic acid sequencing reaction to determine thesequence of the whole or a part of at least one amplified nucleic acidstrand produced in the solid-phase amplification reaction. Theinitiation point for the sequencing reaction may be provided byannealing of a sequencing primer to a product of a solid-phaseamplification reaction. In this connection, one or both of the adaptorsadded during formation of the template library may include a nucleotidesequence which permits annealing of a sequencing primer to amplifiedproducts derived by whole genome or solid-phase amplification of thetemplate library.

The products of solid-phase amplification reactions wherein both forwardand reverse amplification primers are covalently immobilized on thesolid surface are so-called bridged structures formed by annealing ofpairs of immobilized polynucleotide strands and immobilizedcomplementary strands, both strands being attached to the solid supportat the 5′ end. Arrays comprised of such bridged structures provideinefficient templates for typical nucleic acid sequencing techniques,since hybridization of a conventional sequencing primer to one of theimmobilized strands is not favored compared to annealing of this strandto its immobilized complementary strand under standard conditions forhybridization.

In order to provide more suitable templates for nucleic acid sequencing,it may be advantageous to remove or displace substantially all or atleast a portion of one of the immobilized strands in the bridgedstructure in order to generate a template which is at least partiallysingle-stranded. The portion of the template which is single-strandedwill thus be available for hybridization to a sequencing primer. Theprocess of removing all or a portion of one immobilized strand in a‘bridged’ double-stranded nucleic acid structure may be referred toherein as linearization, and is described in further detail in U.S. Pub.2009/0118128, the contents of which are incorporated herein by referencein their entirety.

Bridged template structures may be linearized by cleavage of one or bothstrands with a restriction endonuclease or by cleavage of one strandwith a nicking endonuclease. Other methods of cleavage can be used as analternative to restriction enzymes or nicking enzymes, including interalia chemical cleavage (e.g. cleavage of a diol linkage with periodate),cleavage of abasic sites by cleavage with endonuclease (for example‘USER’, as supplied by NEB, part number M55055), or by exposure to heator alkali, cleavage of ribonucleotides incorporated into amplificationproducts otherwise comprised of deoxyribonucleotides, photochemicalcleavage or cleavage of a peptide linker

Following the cleavage step, regardless of the method used for cleavage,the product of the cleavage reaction may be subjected to denaturingconditions in order to remove the portion(s) of the cleaved strand(s)that are not attached to the solid support. Suitable denaturingconditions, for example sodium hydroxide solution, formamide solution orheat, will be apparent to the skilled reader with reference to standardmolecular biology protocols (Sambrook et al., supra; Ausubel et al.supra). Denaturation results in the production of a sequencing templatewhich is partially or substantially single-stranded. A sequencingreaction may then be initiated by hybridization of a sequencing primerto the single-stranded portion of the template. Thus, the inventionencompasses methods wherein the nucleic acid sequencing reactioncomprises hybridizing a sequencing primer to a single-stranded region ofa linearized amplification product, sequentially incorporating one ormore nucleotides into a polynucleotide strand complementary to theregion of amplified template strand to be sequenced, identifying thebase present in one or more of the incorporated nucleotide(s) andthereby determining the sequence of a region of the template strand.

Another example of a sequencing technology that can be used in themethods of the provided invention is Illumina sequencing. Illuminasequencing workflow is based on three steps: libraries are prepared fromvirtually any nucleic acid sample, amplified to produce clonal clustersand sequenced using massively parallel synthesis. Illumina sequencing isbased on the amplification of DNA on a solid surface using fold-back PCRand anchored primers. Genomic DNA is fragmented, and adapters are addedto the 5′ and 3′ ends of the fragments. DNA fragments that are attachedto the surface of flow cell channels are extended and bridge amplified.Using the methods of the present invention, the joined fragments areattached to the flow cell channels and extended and bridge amplified. Insome embodiments, the linker is removed prior to bridge amplification.In some embodiments, the linker remains attached to the fragments duringamplification. The fragments become double stranded, and the doublestranded molecules are denatured. Multiple cycles of the solid-phaseamplification followed by denaturation can create several millionclusters of approximately 1,000 copies of single-stranded DNA moleculesof the same template in each channel of the flow cell. Primers, DNApolymerase and four fluorophore-labeled, reversibly terminatingnucleotides are used to perform sequential sequencing. After nucleotideincorporation, a laser is used to excite the fluorophores, and an imageis captured and the identity of the first base is recorded. The 3′terminators and fluorophores from each incorporated base are removed andthe incorporation, detection and identification steps are repeated.Sequencing according to this technology is described in U.S. Pat. Nos.7,960,120; 7,835,871; 7,232,656; 7,598,035; 6,911,345; 6,833,246;6,828,100; 6,306,597; 6,210,891; U.S. Pub. 2011/0009278; U.S. Pub.2007/0114362; U.S. Pub. 2006/0292611; and U.S. Pub. 2006/0024681, eachof which are incorporated by reference in their entirety.

Methods of the present invention can be incorporated into the Illuminasequencing platform (commercially available from Illumnia, Inc, SanDiego, CA). Using the present invention, libraries of linked complexescomprising two identical copies of a fragment are prepared and thenattached to the solid support. The complexes are amplified to produceclonal clusters and then sequenced using massively parallel synthesis.In this method, each cluster is seeded with one fragment. With thepresent invention, two identical fragments seed a cluster. Duringsequencing, if there is a lack of agreement at a particular base betweenthe amplicons, the error is detected.

In a preferred embodiment, the joined fragments are attached to the flowcell channel walls. As shown in FIG. 2 , complexes 109 and 119 areattached to a solid support 202, such as a flow cell channel wall.Complex 109 may comprise the sense and complex 119 may comprise theanti-sense. Each complex seeds a cluster. As shown in FIG. 2 , complex109 seeds cluster 1 (205) and complex 119 seeds cluster 2 (207).

FIG. 4A depicts an example of complex 401. Complex 401 comprises alinker 406 and identical copies of a nucleic acid template. However, onecopy of the nucleic acid template comprises an error 410. Complex 401attaches to solid support 402 via binding sites 405. In some examples,the binding sites 405 are complementary oligonucleotides (complementaryto oligonucleotides on the complexes) that are covalently bound to theflow cell surface. As shown in FIG. 4B, complex 401 is extended andbridge amplified to create copies 418. This process is repeated, and asshown in FIG. 4C, a cluster 450 on the solid support 402 forms. Fromthis process, the cluster is a mixture of oligonucleotides derived fromeach half of a complex. About half of the oligonucleotides contain theerror and the other half does not.

FIG. 5A shows two complexes 502 and 503, where complex 502 contains anerror 510. Primers, DNA polymerase and four fluorophore-labeled,reversibly terminating nucleotides are then introduced to performsequential sequencing. After nucleotide incorporation, a laser is usedto excite the fluorophores, and an image is captured and the identity ofthe first base is recorded. Since there is no error at the first base,both bases fluoresce the same. The 3′ terminators and fluorophores fromeach incorporated base are removed and the incorporation, detection andidentification steps are repeated. The steps repeated until the basecontaining the error is reached. At this base, the bases do notfluoresce the same. The bases would fluoresce differently. As shown inFIG. 5B, the mixed fluorescence would indicate that the bases do notmatch. The mixed fluorescence would indicate an error, and the basewould be reported as unknown, or N. See FIG. 5C.

The Illumina Genome Analyzer (detector, commercially available byIllumina) is based on parallel, fluorescence-based readout of millionsof immobilized sequences that are iteratively sequenced using reversibleterminator chemistry. In one example, up to eight DNA libraries arehybridized to an eight-lane flow cell. In each of the lanes,single-stranded library molecules hybridize to complementaryoligonucleotides that are covalently bound to the flow cell surface. Thereverse strand of each library molecule is synthesized and the nowcovalently bound molecule is then further amplified in a process calledbridge amplification. This generates clusters each containing more than1,000 copies of the starting molecule. One strand is then selectivelyremoved, free ends are subsequently blocked and a sequencing primer isannealed onto the adapter sequences of the cluster molecules.

Although the fluorescent imaging system is not sensitive enough todetect the signal from a single template molecule, the detector issensitive to detect the signal from each cluster. In this example of theinvention, the signals from numerous clusters are analyzed. Each clusteris expected to fluoresce at a value, for example, approximate to one ofthe four bases. If the cluster does not fluoresce at a value approximateto one of the four bases, then it is determined that an error exists atthat locus.

After sequencing, images are analyzed and intensities extracted for eachcluster. The Illumina base caller, Bustard, has to handle two effects ofthe four intensity values extracted for each cycle and cluster: first, astrong correlation of the A and C intensities as well as of the G and Tintensities due to similar emission spectra of the fluorophores andlimited separation by the filters used; and second, dependence of thesignal for a specific cycle on the signal of the cycles before andafter, known as phasing and pre-phasing, respectively. Phasing andpre-phasing are caused by incomplete removal of the 3′ terminators andfluorophores, sequences in the cluster missing an incorporation cycle,as well as by the incorporation of nucleotides without effective 3′terminators. Phasing and pre-phasing cause the extracted intensities fora specific cycle to consist of the signal of the current cycle as wellas noise from the preceding and following cycles.

Another example of a sequencing technology that can be used in themethods of the provided invention includes the single molecule,real-time (SMRT) technology of Pacific Biosciences. In SMRT, each of thefour DNA bases is attached to one of four different fluorescent dyes.These dyes are phospholinked. A single DNA polymerase is immobilizedwith a single molecule of template single stranded DNA at the bottom ofa zero-mode waveguide (ZMW). A ZMW is a confinement structure whichenables observation of incorporation of a single nucleotide by DNApolymerase against the background of fluorescent nucleotides thatrapidly diffuse in an out of the ZMW (in microseconds). It takes severalmilliseconds to incorporate a nucleotide into a growing strand. Duringthis time, the fluorescent label is excited and produces a fluorescentsignal, and the fluorescent tag is cleaved off. Detection of thecorresponding fluorescence of the dye indicates which base wasincorporated. The process is repeated. Using methods of the presentinvention, the process is repeated in tandem, with two fragments beinganalyzed.

Another example of a sequencing technique that can be used in themethods of the provided invention is nanopore sequencing (Soni G V andMeller A. (2007) Clin Chem 53: 1996-2001). A nanopore is a small hole,of the order of 1 nanometer in diameter. Immersion of a nanopore in aconducting fluid and application of a potential across it results in aslight electrical current due to conduction of ions through thenanopore. The amount of current which flows is sensitive to the size ofthe nanopore. As a DNA molecule passes through a nanopore, eachnucleotide on the DNA molecule obstructs the nanopore to a differentdegree. Thus, the change in the current passing through the nanopore asthe DNA molecule passes through the nanopore represents a reading of theDNA sequence. Using methods of the present invention, two fragments areanalyzed simultaneously or sequentially, reducing the chance of anerror.

The present invention can be used with nanopore technology, such assingle molecule nanopore-based sequencing by synthesis (Nano-SBS). Thisstrategy can distinguish four bases by detecting 4 different sized tagsreleased from 5′-phosphate-modified nucleotides. As each nucleotide isincorporated into the growing DNA strand during the polymerase reaction,its tag is released and enters a nanopore in release order. Thisproduces a unique ionic current blockade signature due to the tag'sdistinct chemical structure, thereby determining DNA sequenceelectronically at single molecule level with single base resolution.Using the methods of the invention, two identical fragments can beanalyzed simultaneously or sequentially. See Kumar, et al. ScientificReports, Article number 684, doi:10.1038/srep00684.

Functions described above such as sequence read analysis or assembly canbe implemented using systems of the invention that include software,hardware, firmware, hardwiring, or combinations of any of these.

One sequencing method which can be used in accordance with the inventionrelies on the use of modified nucleotides having removable 3′ blocks,for example as described in WO04018497, US 2007/0166705A1 and U.S. Pat.No. 7,057,026, the contents of which are incorporated herein byreference in their entirety. Once the modified nucleotide has beenincorporated into the growing polynucleotide chain complementary to theregion of the template being sequenced there is no free 3′-OH groupavailable to direct further sequence extension and therefore thepolymerase can not add further nucleotides. Once the nature of the baseincorporated into the growing chain has been determined, the 3′ blockmay be removed to allow addition of the next successive nucleotide. Byordering the products derived using these modified nucleotides, it ispossible to deduce the DNA sequence of the DNA template. Such reactionscan be done in a single experiment if each of the modified nucleotideshas a different label attached thereto, known to correspond to theparticular base, to facilitate discrimination between the bases addedduring each incorporation step. Alternatively, a separate reaction maybe carried out containing each of the modified nucleotides separately.

Embodiments of the invention may incorporate modified nucleotides. Themodified nucleotides may be labeled (e.g., fluorescent label) fordetection. Each nucleotide type may thus carry a different fluorescentlabel, for example, as described in U.S. Pub. 2010/0009353, the contentsof which are incorporated herein by reference in their entirety. Thedetectable label need not, however, be a fluorescent label. Any labelcan be used which allows the detection of an incorporated nucleotide.One method for detecting fluorescently labeled nucleotides comprisesusing laser light of a wavelength specific for the labeled nucleotides,or the use of other suitable sources of illumination. The fluorescencefrom the label on the nucleotide may be detected by a CCD camera orother suitable detection means. Suitable instrumentation for recordingimages of clustered arrays is described in W007123744 and U.S. Pub.2010/0111768, the contents of which are incorporated herein by referencein their entirety.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein.

1-20. (canceled)
 21. A method of sequencing a nucleic acid, the methodcomprising: seeding a single sequencing reaction with multiple copies ofa nucleic acid; and conducting the sequencing reaction to determinesequence of the nucleic acid.
 22. The method of claim 21, wherein themultiple copies include first strand copies that are synthesized byextending a polymerase to copy the nucleic acid molecule.
 23. The methodof claim 22, wherein the multiple copies further also include ampliconsmade by copying copies of the nucleic acid molecule.
 24. The method ofclaim 21, wherein the multiple copies of the nucleic molecule representboth senses of a fragment of DNA.
 25. The method of claim 21, furthercomprising, prior to the sequencing step, making the copies of thenucleic acid molecule by annealing each of the linked primers to thenucleic acid molecule, extending the linked primers to make first copiesof the nucleic acid molecule, and copying the first copies with linkedreverse primers to provide a set of amplification templates.
 26. Themethod of claim 25, wherein the linked primer bind to universal bindingsites.
 27. The method of claim 21, further comprising, prior to thesequencing step, making the copies by a process that includes emulsionPCR.
 28. The method of claim 21, further comprising performing bridgeamplification to generate the copies prior to the sequencing step. 29.The method of claim 28, wherein the sequencing generates sequence dataand the method includes detecting, from the sequencing data, polymeraseerror that occurred during the amplification.
 30. The method of claim21, wherein the multiple copies include at least oligonucleotides thatare made by extending a pair of linked primers to copy the nucleic acidmolecule.
 31. The method of claim 30, wherein the two first strandcopies are linked by a linker.
 32. The method of claim 30, wherein thepair of linked primers are linked by a linker that includes polyethyleneglycol (PEG), modified PEG, an inverted base, or a modified base. 33.The method of claim 30, wherein the linked primers include adaptorscompatible with a nucleic acid sequencing system.
 34. A molecularcomplex comprising: a primer extended to include a first copy of anucleic acid fragment; a linker attached to the first primer; and asecond primer attached to the linker, the second primer extended toinclude a second copy of the nucleic acid fragment.
 35. The complex ofclaim 34, wherein the primer and the second primer are each joined tothe linker at a 5′ end.
 36. The complex of claim 34, wherein the linkercomprises polyethylene glycol (PEG), an oligosaccharide, a lipid, ahydrocarbon, a polymer, an antibody, or a protein.
 37. The complex ofclaim 34, wherein the linker comprises DBCO-PEG4, PEG-11, orN-hydroxysuccinimide (NETS) modified PEG.
 38. The complex of claim 34,wherein the linker comprises an inverted or modified base.
 39. Thecomplex of claim 34, wherein the linker does not include guanine,cytosine, adenine, uracil, or thymine.
 40. The complex of claim 34,wherein the first copy of a nucleic acid fragment is further attached toa capture sequence for a sequencing instrument flow cell and the secondcopy of the nucleic acid fragment is further attached to a duplicate ofthe capture sequence.
 41. The complex of claim 40, wherein the complexis immobilized to the flow cell with: the capture sequence annealed to afirst oligonucleotide covalently bound to a surface of the flow cell,and the duplicate of the capture sequence annealed to a secondoligonucleotide covalently bound to a surface of the flow cell.
 42. Thecomplex of claim 40, wherein one of the first or second copy of anucleic acid fragment includes a polymerase error and is not an exactcopy of the nucleic acid fragment.
 43. The complex of claim 42, whereinthe complex is in a cluster of oligonucleotide products attached to theflow cell, the cluster having been produced by bridge amplification fromthe complex, wherein about half of the oligonucleotide products containthe polymerase error.
 44. A method comprising: copying a nucleic acidfragment with two copies of a primer joined by a linker to form acomplex comprising a first copy and a second copy of the nucleic acidfragment joined by the linker.
 45. The method of claim 44, furthercomprising: performing a second copying step with two linked copies of areverse primer that anneals to the first copy or the second copy of thenucleic acid fragment to thereby yield a second complex comprising acopy of the first copy linked to a copy of the second copy.
 46. Themethod of claim 45, wherein the primer and the reverse primer includeadaptors that provide the copy of the first copy and the copy of thesecond copy with universal primer binding sites.
 47. The method of claim46, further comprising performing universal PCR from the universalprimer binding sites with linked pairs of universal primers to generatelinked amplicons.
 48. The method of claim 47, wherein the adaptorsprovide the linked amplicons sequencing primer binding sites.
 49. Themethod of claim 47, further comprising annealing the linked amplicons toa solid support.
 50. The method of claim 47, further comprisingimmobilizing the linked amplicons to the surface of a flow cell bycapturing the linked amplicons to oligonucleotides covalently bound to asurface of the flow cell.
 51. The method of claim 50, wherein eachlinked amplicon includes a first amplified copy and a second amplifiedcopy of the nucleic acid fragment joined by the linker, wherein only oneof the first or second amplified copy of a nucleic acid fragmentincludes a polymerase error and is not an exact copy of the nucleic acidfragment, and the method further comprises performing bridgeamplification to generate a cluster of oligonucleotide products attachedto the flow cell, wherein about half of the oligonucleotide productscontain the polymerase error.
 52. The method of claim 49, furthercomprising sequencing the linked amplicons and identifying amplificationerrors in the first copy or the second copy of the nucleic acidfragment.
 53. The method of claim 44, wherein the linker comprisespolyethylene glycol (PEG), modified PEG, an oligosaccharide, a lipid, ahydrocarbon, a polymer, an antibody, or a protein.